Anti-PDGFR-beta antibodies and uses thereof

ABSTRACT

The present invention provides antibodies that bind to platelet derived growth factor receptor beta (PDGFR-beta) and methods of using the same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human PDGFR-beta with high affinity. The antibodies of the invention are useful for the treatment of diseases and disorders associated with PDGFR-beta signaling and/or PDGFR-beta cellular expression, such as ocular diseases, fibrotic diseases, vascular diseases and cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/992,589 filed Jan. 11, 2016 which application is a divisional of U.S.patent application Ser. No. 14/148,753 filed Jan. 7, 2014, now U.S. Pat.No. 9,265,827 issued Feb. 23, 2016 which application claims the benefitof priority under 35 U.S.C. § 119(e) to U.S. provisional application No.61/750,437, filed on Jan. 9, 2013; 61/863,452, filed on Aug. 8, 2013;and 61/909,421, filed on Nov. 27, 2013, the disclosures of which areherein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for human PDGFR-beta, and methodsof use thereof.

BACKGROUND

Platelet-derived growth factors (PDGFs) are potent mitogens that existas five different dimeric configurations composed of four differentisoform subunits: A, B, C and D. The five dimeric forms of the PDGFs areAA, BB, AB, CC and DD, which are formed by disulfide linkage of thecorresponding individual PDGF monomers. PDGF ligands exert theirbiological effects through their interactions with PDGF receptors(PDGFRs). PDGFRs are single-pass, transmembrane, tyrosine kinasereceptors composed of heterodimeric or homodimeric associations of analpha (a) receptor chain (PDGFR-alpha) and/or a beta (β) receptor chain(PDGFR-beta). Thus, active PDGFRs may consist of αα, ββ or αβ receptorchain pairings. PDGFRs share a common domain structure, including fiveextracellular immunoglobulin (Ig) loops, a transmembrane domain, and asplit intracellular tyrosine kinase (TK) domain. The interaction betweendimeric PDGF ligands and PDGFRs leads to receptor chain dimerization,receptor autophosphorylation and intracellular signal transduction. Ithas been demonstrated in vitro that ββ receptors are activated byPDGF-BB and -DD, while αβ receptors are activated by PDGF-BB, -CC, -DDand -AB, and aa receptors are activated by PDGF-AA, -BB, -CC and -AB(see Andrae et al. (2008) Genes Dev 22 (10):1276-1312).

PDGF signaling has been implicated in various human diseases includingdiseases associated with pathological neovascularization, vascular andfibrotic diseases, tumor growth and eye diseases. Accordingly,inhibitors of PDGF signaling have been suggested for use in a variety oftherapeutic settings. For example, inhibitors of PDGFR-beta have beenproposed for use in treating various diseases and disorders. (Andrae etal. (2008) Genes Dev 22 (10):1276-1312). PDGFR-beta inhibitors includenon-specific small molecule tyrosine kinase inhibitors such as imatinibmesylate, sunitinib malate and CP-673451, as well as anti-PDGFR-betaantibodies (see, e.g., U.S. Pat. Nos. 7,060,271; 5,882,644; 7,740,850;and U.S. Patent Appl. Publ. No. 2011/0177074). Anti-ligand aptamers(e.g., anti-PDGF-B) have also been proposed for therapeuticapplications. Nonetheless, a need exists in the art for new, highlyspecific and potent inhibitors of PDGF signaling.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that bind humanplatelet-derived growth factor receptor beta (“PDGFR-beta”). Theantibodies of the invention are useful, inter alia, for inhibitingPDGFR-beta-mediated signaling and for treating diseases and disorderscaused by or related to PDGFR-beta activity and/or signaling. Theantibodies of the invention are also useful for inducing cell death incells that express high levels of PDGFR-beta on their surfaces.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

The present invention provides antibodies, or antigen-binding fragmentsthereof comprising a heavy chain variable region (HCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 18,34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258,274, 290, 306, and 322, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a light chain variable region (LCVR)having an amino acid sequence selected from the group consisting of SEQID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218,234, 250, 266, 282, 298, 314, and 330, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising a HCVR and LCVR (HCVR/LCVR) sequence pairselected from the group consisting of SEQ ID NO: 2/10, 18/26, 34/42,50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,306/314, and 322/330.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a heavy chain CDR3 (HCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216,232, 248, 264, 280, 296, 312, and 328, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a light chain CDR3 (LCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224,240, 256, 272, 288, 304, 320, and 336, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

In certain embodiments, the antibody or antigen-binding portion of anantibody comprises a HCDR3/LCDR3 amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80,88/96, 104/112, 120/128, 136/144, 152/160, 168/176, 184/192, 200/208,216/224, 232/240, 248/256, 264/272, 280/288, 296/304, 312/320, and328/336.

The present invention also provides an antibody or fragment thereoffurther comprising a heavy chain CDR1 (HCDR1) domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 20,36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260,276, 292, 308, and 324, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity; a heavy chain CDR2 (HCDR2) domain having an amino acidsequence selected from the group consisting of SEQ ID NO: 6, 22, 38, 54,70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294,310, and 326, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;a light chain CDR1 (LCDR1) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 12, 28, 44, 60, 76, 92, 108,124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, and332, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; and a lightchain CDR2 (LCDR2) domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126,142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, and 334, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity.

Certain non-limiting, exemplary antibodies and antigen-binding fragmentsof the invention comprise HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, having the amino acid sequences selected from the groupconsisting of: SEQ ID NOs: 4-6-8-12-14-16 (e.g. H1M3299N);20-22-24-28-30-32 (e.g. H1M3305N); 36-38-40-44-46-48 (e.g. H1M3310N);52-54-56-6062-64 (e.g. H1M3361N); 68-70-72-76-78-80 (e.g. H2M3363N);84-86-88-92-94-96 (e.g. H2M3365N); 100-102-104-108-110-112 (e.g.H2M3368N); 116-118-120-124-126-128 (e.g. H2M3373N);132-134-136-140-142-144 (e.g. H2M3374N); 148-150-152-156-158-160 (e.g.,H4H3094P); 164-166-168-172-174-176 (e.g. H4H3095S);180-182-184-188-190-192 (e.g., H4H3096S); 196-198-200-204-206-208 (e.g.H4H3097S); 212-214-216-220-222-224 (e.g. H4H3098S);228-230-232-236-238-240 (e.g. H4H3099S); 244-246-248-252-254-256 (e.g.H4H3102S); 260-262-264-268-270-272 (e.g. H4H3103S);276-278-280-284-286-288 (e.g. H4H3104S); 292-294-296-300-302-304 (e.g.H4H3105S); 308-310-312-316-318-320 (e.g. H4H3106S); and324-326-328-332-334-336 (e.g. H4H3107S).

In a related embodiment, the invention includes an antibody orantigen-binding fragment of an antibody which specifically bindsPDGFR-beta, wherein the antibody or fragment comprises the heavy andlight chain CDR domains contained within heavy and light chain variableregion (HCVR/LCVR) sequences selected from the group consisting of SEQID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122,130/138, 146/154, 162/170, 178/186, 194/202, 210/218, 226/234, 242/250,258/266, 274/282, 290/298, 306/314, and 322/330. Methods and techniquesfor identifying CDRs within HCVR and LCVR amino acid sequences are wellknown in the art and can be used to identify CDRs within the specifiedHCVR and/or LCVR amino acid sequences disclosed herein. Exemplaryconventions that can be used to identify the boundaries of CDRs include,e.g., the Kabat definition, the Chothia definition, and the AbMdefinition. In general terms, the Kabat definition is based on sequencevariability, the Chothia definition is based on the location of thestructural loop regions, and the AbM definition is a compromise betweenthe Kabat and Chothia approaches. See, e.g., Kabat, “Sequences ofProteins of Immunological Interest”, National Institutes of Health,Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948(1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272(1989). Public databases are also available for identifying CDRsequences within an antibody.

In another aspect, the invention provides nucleic acid moleculesencoding anti-PDGFR-beta antibodies or antigen-binding fragmentsthereof. Recombinant expression vectors carrying the nucleic acids ofthe invention, and host cells into which such vectors have beenintroduced, are also encompassed by the invention, as are methods ofproducing the antibodies by culturing the host cells under conditionspermitting production of the antibodies, and recovering the antibodiesproduced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113,129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, and 321, ora substantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

The present invention also provides an antibody or fragment thereofcomprising a LCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137,153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, and 329, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

The present invention also provides an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 7,23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247,263, 279, 295, 311, and 327, or a substantially identical sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%homology thereof; and a LCDR3 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 15, 31, 47, 63, 79, 95,111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319,and 335, or a substantially identical sequence having at least 90%, atleast 95%, at least 98%, or at least 99% homology thereof.

The present invention also provides an antibody or fragment thereofwhich further comprises a HCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83,99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, and323, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; a HCDR2 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197,213, 229, 245, 261, 277, 293, 309, and 325, or a substantially identicalsequence having at least 90%, at least 95%, at least 98%, or at least99% homology thereof; a LCDR1 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91,107, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315,and 331, or a substantially identical sequence having at least 90%, atleast 95%, at least 98%, or at least 99% homology thereof; and a LCDR2domain encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157,173, 189, 205, 221, 237, 253, 269, 285, 301, 317, and 333, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof.

According to certain embodiments, the antibody or fragment thereofcomprises the heavy and light chain CDR sequences encoded by the nucleicacid sequences of SEQ ID NOs: 1 and 9 (e.g. H1M3299N), 17 and 25 (e.g.H1M3305N), 33 and 41 (e.g. H1M3310N), 49 and 57 (e.g. H1M3361N), 65 and73 (e.g. H2M3363N), 81 and 89 (e.g. H2M3365N), 97 and 105 (e.g.H2M3368N), 113 and 121 (e.g. H2M3373N), 129 and 137 (e.g. H2M3374N), 145and 153 (e.g. H4H3094P), 161 and 169 (e.g. H4H3095S), 177 and 185 (e.g.H4H3096S), 193 and 201 (e.g. H4H3097S), 209 and 217 (e.g. H4H3098S), 225and 233 (e.g. H4H3099S), 241 and 249 (e.g. H4H3102S), 257 and 265 (e.g.H4H3103S), 273 and 281 (e.g. H4H3104S), 289 and 297 (e.g. H4H3105S), 305and 313 (e.g. H4H3106S), or 321 and 329 (e.g. H4H3107S).

The present invention includes anti-PDGFR-beta antibodies having amodified glycosylation pattern. In some applications, modification toremove undesirable glycosylation sites may be useful, or an antibodylacking a fucose moiety present on the oligosaccharide chain, forexample, to increase antibody dependent cellular cytotoxicity (ADCC)function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds PDGFR-beta and a pharmaceutically acceptable carrier.In a related aspect, the invention features a composition which is acombination of an anti-PDGFR-beta antibody and a second therapeuticagent. In one embodiment, the second therapeutic agent is any agent thatis advantageously combined with an anti-PDGFR-beta antibody. Exemplaryagents that may be advantageously combined with an anti-PDGFR-betaantibody include, without limitation, other agents that inhibitPDGFR-beta activity (including other antibodies or antigen-bindingfragments thereof, peptide inhibitors, small molecule antagonists, etc.)and/or agents which do not directly bind PDGFR-beta but nonethelessinterfere with, block or attenuate PDGFR-beta-mediated signaling.Additional combination therapies and co-formulations involving theanti-PDGFR-beta antibodies of the present invention are disclosedelsewhere herein.

In yet another aspect, the invention provides therapeutic methods forinhibiting PDGFR-beta activity using an anti-PDGFR-beta antibody orantigen-binding portion of an antibody of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising an antibody orantigen-binding fragment of an antibody of the invention. The disordertreated is any disease or condition which is improved, ameliorated,inhibited or prevented by removal, inhibition or reduction of PDGFR-betaactivity or signaling. The anti-PDGFR-beta antibodies or antibodyfragments of the invention may function to block the interaction betweenPDGFR-beta and a PDGFR-beta binding partner (e.g., a PDGF ligand), orotherwise inhibit the signaling activity of PDGFR-beta.

The present invention also includes the use of an anti-PDGFR-betaantibody or antigen binding portion of an antibody of the invention inthe manufacture of a medicament for the treatment of a disease ordisorder related to or caused by PDGFR-beta activity in a patient.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a histogram showing the results of a PDGF ligand blockingassay in which PDGFR-beta was captured on a biosensor surface and PDGFligand (BB, DD or AB) was applied to the surface following treatmentwith various anti-PDGFR-beta antibodies of the invention or controlantibody. Results are shown as RUs.

FIG. 2 is a matrix showing the results of an antibody cross-competitionassay in which a first anti-PDGFR-beta antibody (mAb#1) was applied to aPDGFR-beta-coated sensor tip, followed by treatment with a secondanti-PDGFR-beta antibody (mAb#2). Binding responses (numerical values−0.01 to 0.36) for each antibody combination tested are depicted. Lightgrey boxes with black font represent binding response forself-competition. Antibodies competing in both directions, independentof the order of antigen binding, are highlighted in black boxes withwhite font. No competition, suggesting distinct binding regions, isrepresented as white boxes with black font.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about”, when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expressions “platelet-derived growth factor receptor beta”,“PDGFRβ”, “PDGFR-beta”, “PDGFRb” and the like, as used herein, refer tothe human PDGFR-beta protein having the amino acid sequence of SEQ IDNO:341 (see also UniProt accession No. P09619). All references toproteins, polypeptides and protein fragments herein are intended torefer to the human version of the respective protein, polypeptide orprotein fragment unless explicitly specified as being from a non-humanspecies (e.g., “mouse PDGFR-beta”, “monkey PDGFR-beta,” etc.).

As used herein, “an antibody that binds PDGFR-beta” or an“anti-PDGFR-beta antibody” includes antibodies, and antigen-bindingfragments thereof, that bind a soluble fragment of an PDGFR-beta protein(e.g., all or a portion of the extracellular domain of PDGFR-beta)and/or cell surface-expressed PDGFR-beta. The expression “cellsurface-expressed PDGFR-beta” means a PDGFR-beta protein or portionthereof that is expressed on the surface of a cell in vitro or in vivo,such that at least a portion of the PDGFR-beta protein (e.g., aminoacids 33 to 532 of SEQ ID NO:341) is exposed to the extracellular sideof the cell membrane and is accessible to an antigen-binding portion ofan antibody. “Cell surface-expressed PDGFR-beta” includes PDGFR-betamolecules in the context of ββ receptor homodimers as well as PDGFR-betamolecules in the context of αβ heterodimers. Soluble PDGFR-betamolecules include, e.g., monomeric and dimeric PDGFR-beta constructs asdescribed in Example 3 herein (e.g., “PDGFRb.mmh”, SEQ ID NO:337[monomeric], “PDGFRb.mFc”, SEQ ID NO:338 [dimeric] and “PDGFRb.hFc”, SEQID NO:339 [dimeric]), or constructs substantially similar thereto.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., PDGFR-beta). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (CO). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-PDGFR-beta antibody(or antigen-binding portion thereof) may be identical to the humangermline sequences, or may be naturally or artificially modified. Anamino acid consensus sequence may be defined based on a side-by-sideanalysis of two or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment”, asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-PDGFR-beta antibodiesof the invention are human antibodies. The term “human antibody”, asused herein, is intended to include antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody”, as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The present invention includes neutralizing and/or blockinganti-PDGFR-beta antibodies. A “neutralizing” or “blocking” antibody, asused herein, is intended to refer to an antibody whose binding toPDGFR-beta: (i) interferes with the interaction between PDGFR-beta or aPDGFR-beta fragment and a PDGF ligand (e.g., PDGF-BB, PDGF-CC, PDGF-DD,PDGF-AB, etc.); (ii) interferes with the formation of ββ and/or αβreceptor dimers; and/or (ii) results in inhibition of at least onebiological function of PDGFR-beta. The inhibition caused by a PDGFR-betaneutralizing or blocking antibody need not be complete so long as it isdetectable using an appropriate assay. Exemplary assays for detectingPDGFR-beta inhibition are described in the working Examples herein.

The anti-PDGFR-beta antibodies disclosed herein may comprise one or moreamino acid substitutions, insertions and/or deletions in the frameworkand/or CDR regions of the heavy and light chain variable domains ascompared to the corresponding germline sequences from which theantibodies were derived. Such mutations can be readily ascertained bycomparing the amino acid sequences disclosed herein to germlinesequences available from, for example, public antibody sequencedatabases. The present invention includes antibodies, andantigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-PDGFR-beta antibodiescomprising variants of any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includesanti-PDGFR-beta antibodies having HCVR, LCVR, and/or CDR amino acidsequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer,etc. conservative amino acid substitutions relative to any of the HCVR,LCVR, and/or CDR amino acid sequences disclosed herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical”, whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

pH-Dependent Binding

The present invention includes anti-PDGFR-beta antibodies withpH-dependent binding characteristics. For example, an anti-PDGFR-betaantibody of the present invention may exhibit reduced binding toPDGFR-beta at acidic pH as compared to neutral pH. Alternatively,anti-PDGFR-beta antibody of the invention may exhibit enhanced bindingto its antigen at acidic pH as compared to neutral pH. The expression“acidic pH” includes pH values less than about 6.2, e.g., about 6.0,5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35,5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, theexpression “neutral pH” means a pH of about 7.0 to about 7.4. Theexpression “neutral pH” includes pH values of about 7.0, 7.05, 7.1,7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding to PDGFR-beta at acidic pH ascompared to neutral pH” is expressed in terms of a ratio of the K_(D)value of the antibody binding to PDGFR-beta at acidic pH to the K_(D)value of the antibody binding to PDGFR-beta at neutral pH (or viceversa). For example, an antibody or antigen-binding fragment thereof maybe regarded as exhibiting “reduced binding to PDGFR-beta at acidic pH ascompared to neutral pH” for purposes of the present invention if theantibody or antigen-binding fragment thereof exhibits an acidic/neutralK_(D) ratio of about 3.0 or greater. In certain exemplary embodiments,the acidic/neutral K_(D) ratio for an antibody or antigen-bindingfragment of the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5,12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0,60.0, 70.0, 100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Anti-PDGFR-Beta Antibodies Comprising Fc Variants

According to certain embodiments of the present invention,anti-PDGFR-beta antibodies are provided comprising an Fc domaincomprising one or more mutations which enhance or diminish antibodybinding to the FcRn receptor, e.g., at acidic pH as compared to neutralpH. For example, the present invention includes anti-PDGFR-betaantibodies comprising a mutation in the C_(H)2 or a C_(H)3 region of theFc domain, wherein the mutation(s) increases the affinity of the Fcdomain to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0). Such mutations may result in anincrease in serum half-life of the antibody when administered to ananimal. Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/Dor T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Qor K) and/or 434 (e.g., H/F or Y); or a modification at position 250and/or 428; or a modification at position 307 or 308 (e.g., 308F,V308F), and 434. In one embodiment, the modification comprises a 428L(e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g.,V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T,and 256E) modification; a 250Q and 428L modification (e.g., T250Q andM428L); and a 307 and/or 308 modification (e.g., 308F or 308P).

For example, the present invention includes anti-PDGFR-beta antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433Kand N434F). All possible combinations of the foregoing Fc domainmutations, and other mutations within the antibody variable domainsdisclosed herein, are contemplated within the scope of the presentinvention.

Biological Characteristics of the Antibodies

The present invention includes anti-PDGFR-beta antibodies andantigen-binding fragments thereof that bind soluble monomeric or dimericPDGFR-beta molecules with high affinity. For example, the presentinvention includes antibodies and antigen-binding fragments ofantibodies that bind monomeric PDGFR-beta (e.g., at 25° C. or 37° C.)with a K_(D) of less than about 30 nM as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention bind monomeric PDGFR-beta with a K_(D) of lessthan about 25 nM, less than about 20 nM, less than about 15 nM, lessthan about 10 nM, less than about 5 nM, less than about 2 nM, or lessthan about 1 nM, as measured by surface plasmon resonance, e.g., usingthe assay format as defined in Example 3 herein, or a substantiallysimilar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind dimeric PDGFR-beta (e.g., at 25° C. or 37°C.) with a K_(D) of less than about 250 pM as measured by surfaceplasmon resonance, e.g., using the assay format as defined in Example 3herein. In certain embodiments, the antibodies or antigen-bindingfragments of the present invention bind dimeric PDGFR-beta with a K_(D)of less than about 240 pM, less than about 230 pM, less than about 220pM, less than about 210 pM, less than about 200 pM, less than about 190pM, less than about 180 pM, less than about 170 pM, less than about 160pM, less than about 150 pM, less than about 140 pM, less than about 130pM, less than about 120 pM, less than about 110 pM, or less than about100 pM, as measured by surface plasmon resonance, e.g., using the assayformat as defined in Example 3 herein, or a substantially similar assay.

The present invention also includes anti-PDGFR-beta antibodies andantigen-binding fragments thereof that block the binding of one or morePDGF ligand(s) (e.g., PDGF-BB, -AB, -CC, or -DD) to PDGFR-beta. Forexample, the present invention includes anti-PDGFR-beta antibodies thatblock the binding of PDGF-BB to monomeric PDGFR-beta in vitro, with anIC₅₀ value of less than about 300 pM, as measured by an ELISA-basedimmunoassay, e.g., using the assay format as defined in Example 4(A)herein, or a real-time bioassay, e.g., using the assay format as definedin Example 4(B), or a substantially similar assay. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention block the binding of PDGF-BB to monomeric PDGFR-beta in vitrowith an IC₅₀ value of less than about 280 pM, less than about 260 pM,less than about 240 pM, less than about 220 pM, less than about 200 pM,less than about 180 pM, less than about 160 pM, less than about 150 pM,less than about 140 pM, less than about 130 pM, less than about 120 pM,less than about 110 pM, less than about 100 pM, less than about 90 pM,less than about 80 pM, or less than about 75 pM, as measured by anELISA-based immunoassay, e.g., using the assay format as defined inExample 4(A) herein, or a real-time bioassay, e.g., using the assayformat as defined in Example 4(B), or a substantially similar assay.

The present invention also includes anti-PDGFR-beta antibodies andantigen-binding fragments thereof that inhibit PDGF ligand-mediatedactivation of cell surface-expressed PDGFR-beta. For example, thepresent invention includes anti-PDGFR-beta antibodies andantigen-binding fragments thereof that inhibit PDGF-BB- orPDGF-DD-mediated activation of cell surface-expressed PDGFR-beta, withan IC₅₀ value of less than about 500 pM, as measured in a cell-basedblocking bioassay, e.g., using the assay format as defined in Example 6herein, or a substantially similar assay. In certain embodiments, theantibodies or antigen-binding fragments of the present invention blockPDGF-BB- or PDGF-DD-mediated activation of cell surface expressedPDGFR-beta with an IC₅₀ of less than about 400 pM, less than about 350pM, less than about 300 pM, less than about 250 pM, less than about 200pM, less than about 150 pM, less than about 100 pM, less than about 90pM, less than about 80 pM, less than about 70 pM, less than about 60 pM,less than about 50 pM, less than about 40 pM, or less than about 30 pM,as measured in a cell-based blocking bioassay, e.g., using the assayformat as defined in Example 6 herein, or a substantially similar assay.

The present invention also includes anti-PDGFR-beta antibodies andantigen-binding fragments thereof that are internalized into cellsexpressing PDGFR-beta. For example, the present invention includesanti-PDGFR-beta antibodies and antigen-binding fragments thereof thatare effectively internalized into PDGFR-beta-expressing cells asmeasured using a cell-based antibody internalization assay as defined inExample 7 herein, or a substantially similar assay.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combinations thereof.Other biological characteristics of the antibodies of the presentinvention will be evident to a person of ordinary skill in the art froma review of the present disclosure including the working Examplesherein.

Epitope Mapping and Related Technologies

The present invention includes anti-PDGFR-beta antibodies which interactwith one or more amino acids found within the extracellular domain ofhuman PDGFR-beta (e.g., within Ig domains 1, 2, 3, 4 and/or 5 of theextracellular domain of PDGFR-beta). Ig domains 1 through 3 (e.g., aminoacids 1 through 277 of SEQ ID NO:337) are known to be involved in ligandbinding. The present invention includes anti-PDGFR-beta antibodies thatinteract with one or more amino acids found within Ig domain 1 (e.g.,amino acids 1 through 88 of SEQ ID NO:337), Ig domain 2 (e.g., aminoacids 97 through 178 of SEQ ID NO:337) and/or Ig domain 3 (e.g., aminoacids 182 through 277 of SEQ ID NO:337), and thereby effectively blockthe receptor/ligand interaction. In certain exemplary embodiments of thepresent invention, antibodies are provided which specifically interactwith Ig domain 2 (e.g., within amino acids 97 through 178 of SEQ IDNO:337; see, e.g., Example 8). The epitope to which the antibodies bindmay consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) aminoacids located within the extracellular domain of PDGFR-beta.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) located within the extracellulardomain of PDGFR-beta.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-PDGFR-beta antibodies thatbind to the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g. H1M3299N, H1M3305N, H1M3310, H1M3361N, H2M3363N,H2M3365N, H2M3368N, H2M3373N, H2M3374N, H4H3094P, H4H3095S, H4H3096S,H4H3097S, H4H3098S, H4H3099S, H4H3102S, H4H3103S, H4H3104S, H4H3105S,H4H3106S, H4H3107S, etc.). Likewise, the present invention also includesanti-PDGFR-beta antibodies that compete for binding to PDGFR-beta withany of the specific exemplary antibodies described herein (e.g.H1M3299N, H1M3305N, H1M3310N, H1M3361N, H2M3363N, H2M3365N, H2M3368N,H2M3373N, H2M3374N, H4H3094P, H4H3095S, H4H3096S, H4H3097S, H4H3098S,H4H3099S, H4H3102S, H4H3103S, H4H3104S, H4H3105S, H4H3106S, H4H3107S,etc.). For example, the present invention includes anti-PDGFR-betaantibodies that cross-compete for binding to PDGFR-beta with one or moreantibodies of “Bin 1” as defined in Example 5 herein (e.g., H4H3365N,H4H3374N, H4H3103S and H4H3094P). The present invention also includesanti-PDGFR-beta antibodies that cross-compete for binding to PDGFR-betawith one or more antibodies of “Bin 2” as defined in Example 5 herein(e.g., H4H3099S, H4H3107S, H4H3305N and H4H3310N).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-PDGFR-beta antibodyby using routine methods known in the art and exemplified herein. Forexample, to determine if a test antibody binds to the same epitope as areference anti-PDGFR-beta antibody of the invention, the referenceantibody is allowed to bind to a PDGFR-beta protein (e.g., a solubleportion of the PDGFR-beta extracellular domain or cell surface-expressedPDGFR-beta). Next, the ability of a test antibody to bind to thePDGFR-beta molecule is assessed. If the test antibody is able to bind toPDGFR-beta following saturation binding with the referenceanti-PDGFR-beta antibody, it can be concluded that the test antibodybinds to a different epitope than the reference anti-PDGFR-betaantibody. On the other hand, if the test antibody is not able to bind tothe PDGFR-beta molecule following saturation binding with the referenceanti-PDGFR-beta antibody, then the test antibody may bind to the sameepitope as the epitope bound by the reference anti-PDGFR-beta antibodyof the invention. Additional routine experimentation (e.g., peptidemutation and binding analyses) can then be carried out to confirmwhether the observed lack of binding of the test antibody is in fact dueto binding to the same epitope as the reference antibody or if stericblocking (or another phenomenon) is responsible for the lack of observedbinding. Experiments of this sort can be performed using ELISA, RIA,Biacore, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art. In accordance with certainembodiments of the present invention, two antibodies bind to the same(or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excessof one antibody inhibits binding of the other by at least 50% butpreferably 75%, 90% or even 99% as measured in a competitive bindingassay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antibodies are deemed to bind to the same epitope ifessentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies are deemed to have “overlapping epitopes” if onlya subset of the amino acid mutations that reduce or eliminate binding ofone antibody reduce or eliminate binding of the other.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-PDGFR-beta antibody, the above-describedbinding methodology is performed in two orientations: In a firstorientation, the reference antibody is allowed to bind to a PDGFR-betaprotein (e.g., a soluble portion of the PDGFR-beta extracellular domainor cell surface-expressed PDGFR-beta) under saturating conditionsfollowed by assessment of binding of the test antibody to the PDGFR-betamolecule. In a second orientation, the test antibody is allowed to bindto a PDGFR-beta molecule under saturating conditions followed byassessment of binding of the reference antibody to the PDGFR-betamolecule. If, in both orientations, only the first (saturating) antibodyis capable of binding to the PDGFR-beta molecule, then it is concludedthat the test antibody and the reference antibody compete for binding toPDGFR-beta (see, e.g., the assay format described in Example 5 herein,in which soluble PDGFR-beta protein is captured onto sensor tips and thePDGFR-beta-coated sensor tips are treated with a reference antibody[mAb#1] and a test anti-PDGFR-beta antibody [mAb#2] sequentially and inboth bsinding orders). As will be appreciated by a person of ordinaryskill in the art, an antibody that competes for binding with a referenceantibody may not necessarily bind to the same epitope as the referenceantibody, but may sterically block binding of the reference antibody bybinding an overlapping or adjacent epitope.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human PDGFR-beta.

Using VELOCIMMUNE™ technology, for example, or any other known methodfor generating fully human monoclonal antibodies, high affinity chimericantibodies to PDGFR-beta are initially isolated having a human variableregion and a mouse constant region. As in the experimental sectionbelow, the antibodies are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Ifnecessary, mouse constant regions are replaced with a desired humanconstant region, for example wild-type or modified IgG1 or IgG4, togenerate a fully human anti-PDGFR-beta antibody. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region. In certain instances, fully human anti-PDGFR-betaantibodies are isolated directly from antigen-positive B cells.

Bioequivalents

The anti-PDGFR-beta antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies but that retain the ability to bindhuman PDGFR-beta. Such variant antibodies and antibody fragmentscomprise one or more additions, deletions, or substitutions of aminoacids when compared to parent sequence, but exhibit biological activitythat is essentially equivalent to that of the described antibodies.Likewise, the anti-PDGFR-beta antibody-encoding DNA sequences of thepresent invention encompass sequences that comprise one or moreadditions, deletions, or substitutions of nucleotides when compared tothe disclosed sequence, but that encode an anti-PDGFR-beta antibody orantibody fragment that is essentially bioequivalent to ananti-PDGFR-beta antibody or antibody fragment of the invention. Examplesof such variant amino acid and DNA sequences are discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-PDGFR-beta antibodies of the inventionmay be constructed by, for example, making various substitutions ofresidues or sequences or deleting terminal or internal residues orsequences not needed for biological activity. For example, cysteineresidues not essential for biological activity can be deleted orreplaced with other amino acids to prevent formation of unnecessary orincorrect intramolecular disulfide bridges upon renaturation. In othercontexts, bioequivalent antibodies may include anti-PDGFR-beta antibodyvariants comprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention, according to certain embodiments, providesanti-PDGFR-beta antibodies that bind to human PDGFR-beta but not toPDGFR-beta from other species. The present invention also includesanti-PDGFR-beta antibodies that bind to human PDGFR-beta and toPDGFR-beta from one or more non-human species. For example, theanti-PDGFR-beta antibodies of the invention may bind to human PDGFR-betaand may bind or not bind, as the case may be, to one or more of mouse,rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep,cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzeePDGFR-beta. According to certain exemplary embodiments of the presentinvention, anti-PDGFR-beta antibodies are provided which specificallybind human PDGFR-beta (e.g., monomeric and/or dimeric hPDGFR-betaconstructs) and cynomolgus monkey (e.g., Macaca fascicularis) PDGFR-beta(e.g., monomeric and/or dimeric mfPDGFR-beta constructs). (See, e.g.,Example 3, herein).

Immunoconjugates

The invention encompasses anti-PDGFR-beta monoclonal antibodiesconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxic agents include any agent that is detrimental tocells. Examples of suitable cytotoxic agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art, (see for example, WO05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-PDGFR-beta antibodies of thepresent invention can be linked to or co-expressed with anotherfunctional molecule, e.g., another peptide or protein. For example, anantibody or fragment thereof can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody orantibody fragment to produce a bi-specific or a multispecific antibodywith a second binding specificity. For example, the present inventionincludes bi-specific antibodies wherein one arm of an immunoglobulin isspecific for human PDGFR-beta or a fragment thereof, and the other armof the immunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-PDGFR-beta antibodies or antigen-binding fragments thereof of thepresent invention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with PDGFR-beta activity in an adult patient, it maybe advantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administeringanti-PDGFR-beta antibodies may be determined empirically; for example,patient progress can be monitored by periodic assessment, and the doseadjusted accordingly. Moreover, interspecies scaling of dosages can beperformed using well-known methods in the art (e.g., Mordenti et al.,1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing an antibody or other therapeutic protein of the invention,receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol.Chem. 262:4429-4432). The antibodies and other therapeutically activecomponents of the present invention may also be delivered by genetherapy techniques. Methods of introduction include, but are not limitedto, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The composition maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by PDGFR-beta expression, signaling, oractivity, or treatable by blocking the interaction between PDGFR-betaand a PDGFR-beta ligand (e.g., PDGF-BB, PDGF-CC, PDGF-DD, PDGF-AB, etc.)or otherwise inhibiting PDGFR-beta activity and/or signaling. Forexample, the present invention provides methods for treating eyediseases, fibrotic diseases (fibrosis), vascular diseases and/or cancer(tumor growth inhibition) by administering an anti-PDGFR-beta antibody(or pharmaceutical composition comprising an anti-PDGFR-beta antibody)as described herein to a patient in need of such treatment. In thecontext of the methods of treatment described herein, theanti-PDGFR-beta antibody may be administered as a monotherapy (i.e., asthe only therapeutic agent) or in combination with one or moreadditional therapeutic agents (examples of which are described elsewhereherein).

Exemplary eye diseases that are treatable by administering theanti-PDGFR-beta antibodies of the invention include age-related maculardegeneration (e.g., “wet” AMD), exudative AMD, diabetic retinopathy(e.g., proliferative diabetic retinopathy), retinal venous occlusivediseases such as central retinal vein occlusion (CRVO), irisneovascularization, neovascular glaucoma, post-surgical fibrosis inglaucoma, proliferative vitreoretinopathy (PVR), choroidalneovascularization, optic disc neovascularization, cornealneovascularization, retinal neovascularization, vitrealneovascularization, pannus, pterygium, macular edema, diabetic macularedema (DME), vascular retinopathy, retinal degeneration, uveitis, andinflammatory diseases of the eye.

Exemplary fibrotic diseases that are treatable by administering theanti-PDGFR-beta antibodies of the invention include pulmonary fibrosis(e.g., idiopathic pulmonary fibrosis, bleomycin-induced pulmonaryfibrosis, asbestos-induced pulmonary fibrosis, and bronchiolitisobliterans syndrome), chronic asthma, fibrosis associated with acutelung injury and acute respiratory distress (e.g., bacterial pneumoniainduced fibrosis, trauma induced fibrosis, viral pneumonia inducedfibrosis, ventilator induced fibrosis, non-pulmonary sepsis inducedfibrosis and aspiration induced fibrosis), silicosis, radiation-inducedfibrosis, chronic obstructive pulmonary disease (COPD), ocular fibrosis(e.g., ocular fibrotic scarring), skin fibrosis (e.g., scleroderma),hepatic fibrosis (e.g., cirrhosis, alcohol-induced liver fibrosis,non-alcoholic steatohepatitis (NASH), bilary duct injury, primary bilarycirrhosis, infection- or viral-induced liver fibrosis [e.g., chronic HCVinfection], autoimmune hepatitis), kidney (renal) fibrosis, cardiacfibrosis, atherosclerosis, stent restenosis, and myelofibrosis.

Exemplary vascular diseases that are treatable by administering theanti-PDGFR-beta antibodies of the invention include vasoproliferativediseases, pulmonary arterial hypertension, restenosis, vascularscarring, etc.

The present invention also includes methods for treating cancer,inhibiting tumor growth, promoting tumor regression, inhibitingmetastasis, and/or inhibiting pathological angiogenesis (e.g.,angiogenesis related to tumor growth) by administering ananti-PDGFR-beta antibody as described herein to a patient in need ofsuch treatment. For example, the antibodies and antigen-bindingfragments of the present invention may be used to treat, e.g., primaryand/or metastatic tumors arising in the brain and meninges, oropharynx,lung and bronchial tree, gastrointestinal tract, male and femalereproductive tract, muscle, bone, skin and appendages, connectivetissue, spleen, immune system, blood forming cells and bone marrow,liver and urinary tract, and special sensory organs such as the eye. Incertain embodiments, the antibodies and antigen-binding fragments of theinvention are used to treat one or more of the following cancers: renalcell carcinoma, pancreatic carcinoma, breast cancer, head and neckcancer (e.g., cancer of the brain, oral cavity, orophyarynx,nasopharynx, hypopharynx, nasal cavity, paranasal sinuses, larynx, lip,etc.), prostate cancer, urinary bladder cancer, malignant gliomas,osteosarcoma, osteoblastoma, osteochondroma, colorectal cancer, gastriccancer (e.g., gastric cancer with MET amplification), malignantmesothelioma, astrocytoma, glioblastoma, medulloblastoma,retinoblastoma, multiple myeloma, ovarian cancer, small cell lungcancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer,connective tissue neoplasms, Kaposi's sarcoma, basal cell carcinoma,squamous cell carcinoma, or melanoma.

Combination Therapies and Formulations

The present invention includes compositions and therapeutic formulationscomprising any of the anti-PDGFR-beta antibodies described herein incombination with one or more additional therapeutically activecomponents, and methods of treatment comprising administering suchcombinations to subjects in need thereof.

The anti-PDGFR-beta antibodies of the present invention may beco-formulated with and/or administered in combination with, e.g., a VEGFantagonist, e.g., a “VEGF-trap” such as aflibercept or otherVEGF-inhibiting fusion protein as set forth in U.S. Pat. No. 7,087,411,an anti-VEGF antibody or antigen binding fragment thereof (e.g.,bevacizumab, ranibizumab), a small molecule kinase inhibitor of VEGFreceptor (e.g., sunitinib, sorafenib or pazopanib), or an anti-VEGFreceptor antibody. The anti-PDGFR-beta antibody may also be combinedwith a PDGF ligand antagonist (e.g., an anti-PDGF-BB antibody, ananti-PDGF-DD antibody, an anti-PDGF-CC antibody, an anti-PDGF-ABantibody, or other PDGF ligand antagonist such as an aptamer [e.g., ananti-PDGF-B aptamer such as Fovista™, Ophthotech Corp., Princeton,N.J.], an antisense molecule, a ribozyme, an siRNA, a peptibody, ananobody or an antibody fragment directed against a PDGF ligand). Inother embodiments, the anti-PDGFR-beta antibodies of the presentinvention may be co-formulated with and/or administered in combinationwith an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab orpanitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib orerlotinib]), an antagonist of another EGFR family member such asHer2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 or anti-ErbB4antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity),an antagonist specific for EGFRvIII (e.g., an antibody that specificallybinds EGFRvIII), a cMET anagonist (e.g., an anti-cMET antibody), anIGF1R antagonist (e.g., an anti-IGF1R antibody), or a B-raf inhibitor(e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720). In certaininstances, the anti-PDGFR-beta antibodies of the present invention arecombined, co-formulated and/or administered in combination with aPDGFR-alpha inhibitor (e.g., an anti-PDGFR-alpha antibody), a DLL4antagonist (e.g., an anti-DLL4 antibody disclosed in U.S. 2009/0142354such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibodydisclosed in U.S. 2011/0027286 such as H1H685P), etc. Other agents thatmay be beneficially administered in combination with the anti-PDGFR-betaantibodies of the invention include cytokine inhibitors, includingsmall-molecule cytokine inhibitors and antibodies that bind to cytokinessuch as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12,IL-13, IL-17, IL-18, or to their respective receptors.

The anti-PDGFR-beta antibodies of the invention may also be administeredand/or co-formulated in combination with antivirals, antibiotics,analgesics, corticosteroids, steroids, oxygen, antioxidants, metalchelators, IFN-gamma, and/or NSAIDs. The anti-PDGFR-beta antibodies ofthe invention may also be administered as part of a treatment regimenthat also includes radiation treatment and/or conventional chemotherapy(e.g., in the context of methods of treating cancer or inhibiting tumorgrowth).

Any of the aforementioned additional therapeutically active componentsmay be administered in combination with any of the anti-PDGFR-betaantibodies of the present invention for the treatment of any disease ordisorder in which administration of an anti-PDGFR-beta antibody isbeneficial, including, e.g., any of the eye diseases, fibrotic diseases,vascular diseases and/or cancers mentioned herein. For example, in thecontext of treating an eye disease (e.g., wet AMD, diabetic retinopathy,CRVO, or any of the other eye diseases described herein), ananti-PDGFR-beta antibody of the present invention may be co-formulatedwith, and/or administered in combination with a VEGF antagonist, e.g., a“VEGF-trap” such as aflibercept or other VEGF-inhibiting fusion proteinas set forth in U.S. Pat. No. 7,087,411, or an anti-VEGF antibody orantigen binding fragment thereof (e.g., bevacizumab, or ranibizumab).

In exemplary embodiments in which an anti-PDGFR-beta antibody of theinvention is administered in combination with a VEGF antagonist (e.g., aVEGF trap such as aflibercept), including administration ofco-formulations comprising an anti-PDGFR-beta antibody and a VEGFantagonist, the individual components may be administered to a subjectand/or co-formulated using a variety of dosage combinations. Forexample, the anti-PDGFR-beta antibody may be administered to a subjectand/or contained in a co-formulation in an amount selected from thegroup consisting of 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5mg, 4.0 mg, 4.5 mg, 5.0 mg, and 5.5 mg; and the VEGF antagonist (e.g., aVEGF trap such as aflibercept) may be administered to the subject and/orcontained in a co-formulation in an amount selected from the groupconsisting of 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg,1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg,2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg and 3.0 mg. Exemplary anti-PDGFR-betaantibody/aflibercept dosage combinations of the present inventioninclude, e.g.: (i) 0.2 mg anti-PDGFR-beta antibody +2 mg aflibercept;(ii) 0.5 mg anti-PDGFR-beta antibody +2 mg aflibercept; (iii) 1 mganti-PDGFR-beta antibody +2 mg aflibercept; (iv) 3 mg anti-PDGFR-betaantibody +2 mg aflibercept; and (v) 4 mg anti-PDGFR-beta antibody +2 mgaflibercept. The combinations/co-formulations may be administered to asubject according to any of the administration regimens disclosedelsewhere herein, including, e.g., once every week, once every 2 weeks,once every 3 weeks, once every month, once every 2 months, once every 3months, once every 4 months, once every 5 months, once every 6 months,etc.

The additional therapeutically active component(s) may be administeredto a subject prior to administration of an anti-PDGFR-beta antibody ofthe present invention. For example, a first component may be deemed tobe administered “prior to” a second component if the first component isadministered 1 week before, 72 hours before, 60 hours before, 48 hoursbefore, 36 hours before, 24 hours before, 12 hours before, 6 hoursbefore, 5 hours before, 4 hours before, 3 hours before, 2 hours before,1 hour before, 30 minutes before, 15 minutes before, 10 minutes before,5 minutes before, or less than 1 minute before administration of thesecond component. In other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject afteradministration of an anti-PDGFR-beta antibody of the present invention.For example, a first component may be deemed to be administered “after”a second component if the first component is administered 1 minuteafter, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutesafter, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5hours after, 6 hours after, 12 hours after, 24 hours after, 36 hoursafter, 48 hours after, 60 hours after, 72 hours after administration ofthe second component. In yet other embodiments, the additionaltherapeutically active component(s) may be administered to a subjectconcurrent with administration of an anti-PDGFR-beta antibody of thepresent invention. “Concurrent” administration, for purposes of thepresent invention, includes, e.g., administration of an anti-PDGFR-betaantibody and an additional therapeutically active component to a subjectin a single dosage form (e.g., co-formulated), or in separate dosageforms administered to the subject within about 30 minutes or less ofeach other. If administered in separate dosage forms, each dosage formmay be administered via the same route (e.g., both the anti-PDGFR-betaantibody and the additional therapeutically active component may beadministered intravitreally, subcutaneously, etc.); alternatively, eachdosage form may be administered via a different route (e.g., theanti-PDGFR-beta antibody may be administered Intravitreally, and theadditional therapeutically active component may be administeredsystemically). In any event, administering the components in a singledosage from, in separate dosage forms by the same route, or in separatedosage forms by different routes are all considered “concurrentadministration”, for purposes of the present disclosure. For purposes ofthe present disclosure, administration of an anti-PDGFR-beta antibody“prior to”, “concurrent with”, or “after” (as those terms are definedherein above) administration of an additional therapeutically activecomponent is considered administration of an anti-PDGFR-beta antibody“in combination with” an additional therapeutically active component).

The present invention includes pharmaceutical compositions in which ananti-PDGFR-beta antibody of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

The present invention also includes additional therapeutic compositionscomprising a combination of a PDGF antagonist and a VEGF antagonist.PDGF antagonists according to this aspect of the invention include PDGFreceptor antagonists as well as PDGF ligand antagonists. Likewise, VEGFantagonists according to this aspect of the invention include VEGFreceptor antagonists as well as VEGF ligand antagonists.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an anti-PDGFR-beta antibody (or a pharmaceutical compositioncomprising a combination of an anti-PDGFR-beta antibody and any of theadditional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an anti-PDGFR-beta antibodyof the invention. As used herein, “sequentially administering” meansthat each dose of anti-PDGFR-beta antibody is administered to thesubject at a different point in time, e.g., on different days separatedby a predetermined interval (e.g., hours, days, weeks or months). Thepresent invention includes methods which comprise sequentiallyadministering to the patient a single initial dose of an anti-PDGFR-betaantibody, followed by one or more secondary doses of the anti-PDGFR-betaantibody, and optionally followed by one or more tertiary doses of theanti-PDGFR-beta antibody.

The terms “initial dose”, “secondary doses”, and “tertiary doses”, referto the temporal sequence of administration of the anti-PDGFR-betaantibody of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-PDGFR-beta antibody, but generally may differ from one another interms of frequency of administration. In certain embodiments, however,the amount of anti-PDGFR-beta antibody contained in the initial,secondary and/or tertiary doses varies from one another (e.g., adjustedup or down as appropriate) during the course of treatment. In certainembodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered atthe beginning of the treatment regimen as “loading doses” followed bysubsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose”, as used herein, means, in a sequence of multipleadministrations, the dose of anti-PDGFR-beta antibody which isadministered to a patient prior to the administration of the very nextdose in the sequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-PDGFR-beta antibody. For example, in certain embodiments,only a single secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens in which 2 to 6loading doses are administered to a patient at a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the invention, if the loading doses areadministered at a frequency of, e.g., once a month (e.g., two, three,four, or more loading doses administered once a month), then themaintenance doses may be administered to the patient once every fiveweeks, once every six weeks, once every seven weeks, once every eightweeks, once every ten weeks, once every twelve weeks, etc.).

Diagnostic Uses of the Antibodies

The anti-PDGFR-beta antibodies of the present invention may also be usedto detect and/or measure PDGFR-beta, or PDGFR-beta-expressing cells in asample, e.g., for diagnostic purposes. For example, an anti-PDGFR-betaantibody, or fragment thereof, may be used to diagnose a condition ordisease characterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of PDGFR-beta. Exemplarydiagnostic assays for PDGFR-beta may comprise, e.g., contacting asample, obtained from a patient, with an anti-PDGFR-beta antibody of theinvention, wherein the anti-PDGFR-beta antibody is labeled with adetectable label or reporter molecule. Alternatively, an unlabeledanti-PDGFR-beta antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure PDGFR-beta in asample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in PDGFR-beta diagnostic assays according tothe present invention include any tissue or fluid sample obtainable froma patient which contains detectable quantities of PDGFR-beta protein, orfragments thereof, under normal or pathological conditions. Generally,levels of PDGFR-beta in a particular sample obtained from a healthypatient (e.g., a patient not afflicted with a disease or conditionassociated with abnormal PDGFR-beta levels or activity) will be measuredto initially establish a baseline, or standard, level of PDGFR-beta.This baseline level of PDGFR-beta can then be compared against thelevels of PDGFR-beta measured in samples obtained from individualssuspected of having a PDGFR-beta related disease or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Human Antibodies to PDGFR-Beta

An immunogen comprising the PDGFR-beta ecto domain was administereddirectly, with an adjuvant to stimulate the immune response, to aVELOCIMMUNE® mouse comprising DNA encoding human Immunoglobulin heavyand kappa light chain variable regions. The antibody immune response wasmonitored by a PDGFR-beta-specific immunoassay. When a desired immuneresponse was achieved splenocytes were harvested and fused with mousemyeloma cells to preserve their viability and form hybridoma cell lines.The hybridoma cell lines were screened and selected to identify celllines that produce PDGFR-beta-specific antibodies. Using this techniqueseveral anti-PDGFR-beta chimeric antibodies (i.e., antibodies possessinghuman variable domains and mouse constant domains) were obtained;exemplary antibodies generated in this manner were designated asfollows: H1M3299N, H1M3305N, H1M3310N, H1M3361N, H2M3363N, H2M3365N,H2M3368N, H2M3373N and H2M3374N. The human variable domains from thechimeric antibodies were subsequently cloned onto human constant domainsto make fully human anti-PDGFR-beta antibodies as described herein.

Anti-PDGFR-beta antibodies were also isolated directly fromantigen-positive B cells without fusion to myeloma cells, as describedin U.S. 2007/0280945A1. Using this method, several fully humananti-PDGFR-beta antibodies (i.e., antibodies possessing human variabledomains and human constant domains) were obtained; exemplary antibodiesgenerated in this manner were designated as follows: H4H3394P, H4H3095S,H4H3096S, H4H3097S, H4H3098S, H4H3099S, H4H3102S, H4H3103S, H4H3104S,H4H3105S, H4H3106S, H4H3107S.

Certain biological properties of the exemplary anti-PDGFR-betaantibodies generated in accordance with the methods of this Example aredescribed in detail in the Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs of selected anti-PDGFR-beta antibodies and theircorresponding antibody identifiers.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 3299N 2 4 6 8 10 12 14 16 3305N 18 20 22 24 26 28 3032 3310N 34 36 38 40 42 44 46 48 3361N 50 52 54 56 58 60 62 64 3363N 6668 70 72 74 76 78 80 3365N 82 84 86 88 90 92 94 96 3368N 98 100 102 104106 108 110 112 3373N 114 116 118 120 122 124 126 128 3374N 130 132 134136 138 140 142 144 3094P 146 148 150 152 154 156 158 160 3095S 162 164166 168 170 172 174 176 3096S 178 180 182 184 186 188 190 192 3097S 194196 198 200 202 204 206 208 3098S 210 212 214 216 218 220 222 224 3099S226 228 230 232 234 236 238 240 3102S 242 244 246 248 250 252 254 2563103S 258 260 262 264 266 268 270 272 3104S 274 276 278 280 282 284 286288 3105S 290 292 294 296 298 300 302 304 3106S 306 308 310 312 314 316318 320 3107S 322 324 326 328 330 332 334 336

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M”, “H2M”, “H4H”), followed by anumerical identifier (e.g. “3299”, “3363”, or “3094” as shown in Table1), followed by a “P,” “N” or “S” suffix. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H1M3299N”, “H2M3363N”, “H4H3094”, etc. The H1M, H2M and H4H prefixes onthe antibody designations used herein indicate the particular Fc regionisotype of the antibody. For example, an “H1M” antibody has a mouse IgG1Fc, whereas an “H4H” antibody has a human IgG4 Fc. As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Table 1—will remain the same, and the bindingproperties are expected to be identical or substantially similarregardless of the nature of the Fc domain.

Control Construct Used in the Following Examples

An anti-PDGFR-beta control antibody was included in the followingExamples for comparative purposes. The control antibody is designatedherein as Control I: a human anti-PDGFR-beta antibody with heavy andlight chain variable domain sequences of “2C5” as set forth in U.S. Pat.No. 7,740,850.

Example 3. Antibody Binding to Human PDGFR-Beta as Determined by SurfacePlasmon Resonance

Binding affinities and kinetic constants for antigen binding to selectedpurified anti-human PDGFR-beta monoclonal antibodies were determinedusing a real-time surface plasmon resonance biosensor (Biacore T100, GEHealthcare Life Sciences, Piscataway, N.J.) assay at 25° C. and 37° C.Antibodies, expressed as either mouse Fc (prefix H1M; H2M) or human Fc(prefix H4H), were captured on their respective anti-Fc sensor surfaces(Mab capture format). Different concentrations of soluble monomericPDGFR-beta constructs (hPDGFRb.mmh [SEQ ID NO:337], Macaca fascicularisPDGFRb.mmh [SEQ ID NO:340]) or dimeric PDGFR-beta constructs (humanPDGFRb.mFc [SEQ ID NO:338] or human PDGFRb.hFc [SEQ ID NO:339]) wereinjected over the anti-PDFR-beta monoclonal antibody captured surface ata flow rate of 50 μL/min. Kinetic association (k_(a)) and dissociation(k_(d)) rate constants were determined by processing and fitting thedata to a 1:1 binding model using Scrubber 2.0 curve fitting software.Binding dissociation equilibrium constants (K_(D)) and dissociativehalf-lives (t_(1/2)) were calculated from the kinetic rate constants as:K_(D) (M)=k_(d)/k_(a); and t_(1/2) (min=(In2/(60*k_(d)). Kinetic bindingparameters for different anti-PDGFR-beta monoclonal antibodies are shownin Tables 2 to 5. (NB=no binding observed under the conditions used;NT=not tested).

TABLE 2 Binding Characteristics of Anti-PDGFR-beta Antibodies (Mouse FcFormat) to Monomeric and Dimeric PDGFR-beta constructs at 25° C.Antibody Analyte ka (Ms⁻¹) kd (s⁻¹) K_(D) (Molar) t_(1/2) (min) H1M3305NhPDGFRb.mmh 3.12E+04 2.52E−05 8.08E−10 458 mfPDGFRb.mmh 5.10E+044.16E−05 8.16E−10 278 hPDGFRb.mFc 1.62E+05 1.00E−06 6.18E−12 11550H1M3310N hPDGFRb.mmh 1.40E+04 1.00E−06 7.00E−11 11550 mfPDGFRb.mmh1.00E+04 1.00E−06 2.00E−10 11550 hPDGFRb.mFc 1.27E+04 1.00E−06 7.89E−1111550 H1M3299N hPDGFRb.mmh 2.11E+04 9.20E−04 4.35E−08 13 mfPDGFRb.mmh NBNB NB NB hPDGFRb.mFc 2.59E+04 1.65E−04 6.35E−09 70 H1M3361N hPDGFRb.mmh1.73E+05 1.26E−03 7.29E−09 9 mfPDGFRb.mmh 1.00E+04 3.89E−05 3.90E−09 297hPDGFRb.mFc 1.31E+04 1.00E−06 7.65E−11 11550 H2M3363N hPDGFRb.mmh7.11E+04 3.33E−03 4.68E−08 3 mfPDGFRb.mmh 5.00E+04 6.85E−05 1.40E−09 169hPDGFRb.mFc 1.04E+05 4.03E−06 3.86E−11 2867 H2M3365N hPDGFRb.mmh4.54E+04 1.27E−04 2.79E−09 91 mfPDGFRb.mmh 6.00E+04 2.06E−04 3.40E−09 56hPDGFRb.mFc 2.36E+05 8.01E−05 3.40E−10 144 H2M3368N hPDGFRb.mmh 4.61E+043.41E−04 7.41E−09 34 mfPDGFRb.mmh 7.00E+03 1.85E−04 3.00E−08 63hPDGFRb.mFc 1.18E+05 3.70E−05 3.13E−10 313 H2M3373N hPDGFRb.mmh 1.89E+052.35E−03 1.24E−08 5 mfPDGFRb.mmh 1.30E+05 2.38E−03 1.83E−08 5hPDGFRb.mFc 4.73E+05 2.40E−04 5.07E−10 48 H1M3374N hPDGFRb.mmh 1.67E+053.31E−04 1.99E−09 35 mfPDGFRb.mmh 1.37E+05 3.71E−04 2.70E−09 31hPDGFRb.mFc 9.96E+05 1.07E−04 1.08E−10 108

TABLE 3 Binding Characteristics of Anti-PDGFR-beta Antibodies (Human FcFormat) to Monomeric and Dimeric PDGFR-beta constructs at 25° C.Antibody Analyte ka (Ms⁻¹) kd (s⁻¹) K_(D) (Molar) t_(1/2) (min) H4H3305NhPDGFRb.mmh 5.99E+04 1.09E−04 1.81E−09 106 mfPDGFRb.mmh 6.12E+041.11E−04 1.82E−09 104 hPDGFRb.hFc 1.38E+05 3.42E−05 2.48E−10 338H4H3310N hPDGFRb.mmh 2.61E+04 8.92E−05 3.41E−09 130 mfPDGFRb.mmh2.88E+04 1.08E−04 3.75E−09 107 hPDGFRb.hFc 4.45E+04 2.90E−05 6.52E−10398 H4H3365N hPDGFRb.mmh 8.53E+04 1.42E−04 1.66E−09 81 mfPDGFRb.mmh8.83E+04 1.50E−04 1.70E−09 77 hPDGFRb.hFc 1.84E+05 4.55E−05 2.44E−10 254H4H3374N hPDGFRb.mmh 2.83E+05 3.58E−04 1.26E−09 32 mfPDGFRb.mmh 2.84E+054.72E−04 1.66E−09 24 hPDGFRb.hFc 6.00E+05 8.93E−05 1.48E−10 129 H4H3107ShPDGFRb.mmh 2.21E+05 1.91E−04 8.63E−10 61 mfPDGFRb.mmh 2.36E+05 1.98E−048.36E−10 58 hPDGFRb.hFc 5.29E+05 4.24E−05 8.01E−11 272 H4H3102ShPDGFRb.mmh 5.09E+05 4.55E−04 8.90E−10 25 mfPDGFRb.mmh 2.83E+05 4.89E−041.73E−09 24 hPDGFRb.hFc 3.00E+05 1.18E−04 3.90E−10 98 H4H3099ShPDGFRb.mmh 1.45E+05 1.69E−04 1.16E−09 68 mfPDGFRb.mmh 1.66E+05 1.64E−049.87E−10 71 hPDGFRb.hFc 2.38E+05 5.48E−05 2.30E−10 211 H4H3098ShPDGFRb.mmh 3.86E+05 5.96E−04 1.54E−09 19 mfPDGFRb.mmh 1.36E+05 9.40E−036.89E−08 1.2 hPDGFRb.hFc 2.80E+05 6.22E−05 2.19E−10 186 H4H3104ShPDGFRb.mmh 4.28E+05 6.88E−04 1.61E−09 17 mfPDGFRb.mmh 7.86E+05 7.14E−049.09E−10 16 hPDGFRb.hFc 4.80E+05 1.46E−04 3.04E−10 79 H4H3094PhPDGFRb.mmh 1.65E+05 2.57E−04 1.56E−09 45 mfPDGFRb.mmh 1.77E+05 2.89E−041.63E−09 40 hPDGFRb.hFc 2.42E+05 6.20E−05 2.56E−10 186 H4H3103ShPDGFRb.mmh 3.35E+05 1.05E−03 3.13E−09 11 mfPDGFRb.mmh 3.59E+05 1.16E−033.24E−09 10 hPDGFRb.hFc 6.21E+05 1.64E−04 2.64E−10 70 H4H3106ShPDGFRb.mmh 2.99E+05 7.44E−04 2.49E−09 16 mfPDGFRb.mmh 1.90E+05 8.82E−044.65E−09 13 hPDGFRb.hFc 3.14E+05 2.15E−04 6.86E−10 54 H4H3105ShPDGFRb.mmh 2.46E+05 7.84E−04 3.19E−09 15 mfPDGFRb.mmh 1.80E+05 9.32E−045.20E−09 12 hPDGFRb.hFc 2.47E+05 2.25E−04 9.10E−10 51 H4H3095ShPDGFRb.mmh 2.85E+05 1.36E−03 4.78E−09 8 mfPDGFRb.mmh 2.07E+05 1.75E−038.50E−09 7 hPDGFRb.hFc 3.21E+05 2.32E−04 7.20E−10 50 H4H3096ShPDGFRb.mmh 2.81E+05 1.04E−03 3.68E−09 11 mfPDGFRb.mmh 1.82E+05 1.17E−036.39E−09 10 hPDGFRb.hFc 2.22E+05 2.60E−04 1.17E−09 44 H4H3097ShPDGFRb.mmh NB NB NB NB mfPDGFRb.mmh NB NB NB NB hPDGFRb.hFc NB NB NB NBControl I hPDGFRb.mmh 2.77E+05 3.49E−03 1.26E−08 3 mfPDGFRb.mmh 3.02E+052.43E−03 8.06E−09 5 hPDGFRb.hFc 5.39E+05 1.50E−04 2.78E−10 77

TABLE 4 Binding Characteristics of Anti-PDGFR-beta Antibodies (Mouse FcFormat) to Monomeric and Dimeric PDGFR-beta constructs at 37° C.Antibody Analyte ka (Ms⁻¹) kd (s⁻¹) K_(D) (Molar) t_(1/2) (min) H1M3305NhPDGFRb.mmh 1.16E+05 1.02E−04 8.80E−10 113 mfPDGFRb.mmh NT NT NT NThPDGFRb.mFc NT NT NT NT H1M3310N hPDGFRb.mmh 3.53E+04 6.46E−05 1.83E−09179 mfPDGFRb.mmh NT NT NT NT hPDGFRb.mFc NT NT NT NT H1M3299NhPDGFRb.mmh 3.16E+04 2.17E−03 6.86E−08 5 mfPDGFRb.mmh NT NT NT NThPDGFRb.mFc NT NT NT NT H1M3361N hPDGFRb.mmh 3.04E+05 8.33E−03 2.74E−081.4 mfPDGFRb.mmh NT NT NT NT hPDGFRb.mFc NT NT NT NT H2M3363NhPDGFRb.mmh 2.86E+05 5.03E−03 1.76E−08 2 mfPDGFRb.mmh NT NT NT NThPDGFRb.mFc NT NT NT NT H2M3365N hPDGFRb.mmh 1.15E+05 5.51E−04 4.79E−0921 mfPDGFRb.mmh NT NT NT NT hPDGFRb.mFc NT NT NT NT H2M3368N hPDGFRb.mmh1.37E+05 8.44E−04 6.17E−09 14 mfPDGFRb.mmh NT NT NT NT hPDGFRb.mFc NT NTNT NT H2M3373N hPDGFRb.mmh 4.10E+05 1.22E−02 2.98E−08 0.9 mfPDGFRb.mmhNT NT NT NT hPDGFRb.mFc NT NT NT NT H1M3374N hPDGFRb.mmh 4.63E+057.90E−04 1.71E−09 15 mfPDGFRb.mmh NT NT NT NT hPDGFRb.mFc NT NT NT NT

TABLE 5 Binding Characteristics of Anti-PDGFR-beta Antibodies (Human FcFormat) to Monomeric and Dimeric PDGFR-beta constructs at 37° C.Antibody Analyte ka (Ms⁻¹) kd (s⁻¹) K_(D) (Molar) t_(1/2) (min) H4H3305NhPDGFRb.mmh 1.84E+05 3.55E−04 1.93E−09 33 mfPDGFRb.mmh 1.91E+05 3.90E−042.04E−09 30 hPDGFRb.hFc 2.47E+05 4.85E−05 1.97E−10 238 H4H3310NhPDGFRb.mmh 5.09E+04 3.39E−04 6.65E−09 34 mfPDGFRb.mmh 5.14E+04 3.92E−047.62E−09 29 hPDGFRb.hFc 7.13E+04 4.50E−05 6.32E−10 256 H4H3365NhPDGFRb.mmh 1.90E+05 1.02E−03 5.38E−09 11 mfPDGFRb.mmh 2.00E+05 1.01E−035.06E−09 11 hPDGFRb.hFc 2.50E+05 2.64E−04 1.05E−09 44 H4H3374NhPDGFRb.mmh 6.85E+05 1.26E−03 1.84E−09 9 mfPDGFRb.mmh 6.70E+05 1.77E−032.63E−09 7 hPDGFRb.hFc 1.63E+06 2.91E−04 1.78E−10 40 H4H3107ShPDGFRb.mmh 6.05E+05 8.79E−04 1.45E−09 13 mfPDGFRb.mmh 6.83E+05 9.42E−041.38E−09 12 hPDGFRb.hFc 6.95E+05 1.15E−04 1.65E−10 101 H4H3102ShPDGFRb.mmh 1.04E+06 1.47E−03 1.42E−09 8 mfPDGFRb.mmh 5.74E+05 1.64E−032.86E−09 7 hPDGFRb.hFc 4.20E+05 3.19E−04 7.60E−10 36 H4H3099ShPDGFRb.mmh 2.67E+05 6.39E−04 2.39E−09 18 mfPDGFRb.mmh 3.00E+05 6.52E−042.17E−09 18 hPDGFRb.hFc 5.40E+05 1.05E−04 1.93E−10 110 H4H3098ShPDGFRb.mmh 7.33E+05 1.71E−03 2.34E−09 7 mfPDGFRb.mmh 2.80E+05 2.67E−029.56E−08 0.4 hPDGFRb.hFc 3.74E+05 7.66E−05 2.06E−10 151 H4H3104ShPDGFRb.mmh 8.33E+05 2.80E−03 3.37E−09 4 mfPDGFRb.mmh 7.40E+05 2.99E−034.05E−09 4 hPDGFRb.hFc 9.36E+05 5.67E−04 6.06E−10 20 H4H3094PhPDGFRb.mmh 2.23E+05 1.47E−03 6.58E−09 8 mfPDGFRb.mmh 2.53E+05 1.70E−036.69E−09 7 hPDGFRb.hFc 2.83E+05 2.48E−04 8.77E−10 47 H4H3103ShPDGFRb.mmh 4.92E+05 4.97E−03 1.01E−08 2 mfPDGFRb.mmh 5.44E+05 5.56E−031.02E−08 2 hPDGFRb.hFc 7.57E+05 3.06E−04 4.05E−10 38 H4H3106ShPDGFRb.mmh 3.94E+05 3.35E−03 8.49E−09 3 mfPDGFRb.mmh 3.72E+05 3.45E−039.26E−09 3 hPDGFRb.hFc 3.56E+05 7.41E−04 2.08E−09 16 H4H3105ShPDGFRb.mmh 3.14E+05 3.54E−03 1.13E−08 3 mfPDGFRb.mmh 2.89E+05 4.16E−031.44E−08 3 hPDGFRb.hFc 2.80E+05 8.24E−04 3.00E−09 14 H4H3095ShPDGFRb.mmh 4.52E+05 6.24E−03 1.38E−08 2 mfPDGFRb.mmh 2.39E+05 7.97E−033.33E−08 1.5 hPDGFRb.hFc 4.25E+05 7.10E−04 1.67E−09 16 H4H3096ShPDGFRb.mmh 4.52E+05 6.24E−03 1.38E−08 2 mfPDGFRb.mmh 1.62E+05 5.12E−033.16E−08 2 hPDGFRb.hFc 2.50E+05 7.93E−04 3.10E−09 15 H4H3097ShPDGFRb.mmh NB NB NB NB mfPDGFRb.mmh NB NB NB NB hPDGFRb.hFc NB NB NB NBControl I hPDGFRb.mmh 4.50E+05 1.46E−02 3.25E−08 0.8 mfPDGFRb.mmh4.89E+05 9.82E−03 2.01E−08 1.2 hPDGFRb.hFc 8.04E+05 2.17E−04 2.70E−10 53

As shown in Tables 2-5, Several anti-PDGFR-beta antibodies of thepresent invention displayed sub-nanomolar affinity to the human and M.fascicularis PDGFR-beta constructs. In addition, several clones showedtighter (lower K_(D)) binding to the PDGFR-beta constructs than thereference (Control 1) antibody.

Example 4. Anti-PDGFR-Beta Antibodies Block Binding of PDGF Ligands toPDGFR-Beta

A. Receptor/Ligand Blocking Assessed Using an ELISA-Based Immunoassay

The ability of certain anti-human PDGFR-beta antibodies of the inventionto block receptor binding to its ligand PDGF-BB was first evaluated withan ELISA-based immunoassay. Briefly, plates were coated with humanPDGF-BB (2 μg/mL). Separately, 250 pM of biotinylated solublehPDGFR-beta.mmh (“biot-hPDGFR-beta-mmh”, SEQ ID NO:337) was premixedwith serially diluted anti-PDGFR-beta antibodies (0-100 nM) for 1 hr atroom temperature (25° C.). The equilibrated PDGFR-beta/antibodysolutions were added to ligand-coated plates, allowed to incubate for 1hr, and washed. Levels of bound biot-hPDGFR-beta.mmh were detected usingHRP conjugated streptavidin. Data were analyzed using Prism software andIC₅₀ values were calculated as the amount of antibody required toachieve 50% reduction of hPDGFR-beta-mmh bound to ligand. Maximumblocking values were also calculated and reflect the ability of theantibody to block relative to baseline. The absorbance measured at theconstant amount of 250 pM biot-hPDGFR-beta-mmh on the dose curve isdefined as 0% blocking and the absorbance with no added PDGFR-beta isdefined as 100%. The absorbance of the wells containing the highestantibody concentration determined the maximum blocking percent. Resultsare shown in Table 6. (“E” indicates that the antibody is an enhancer,i.e., signal was higher in the presence of some concentrations of theantibody than in the absence of the antibody.)

TABLE 6 Anti-PDGFR-beta Antibody Blocking of PDGF-BB Binding toPDGFR-beta IC₅₀ of Antibody Blocking of Ligand/Receptor % MaximumAntibody Interaction (Molar) Blocking H1M3299N 7.6E−09 67 H1M3305N8.5E−11 83 H1M3310N 1.2E−10 88 H1M3361N 1.0E−10 76 H1M3374N 7.7E−11 88H2M3363N 4.1E−09 77 H2M3365N 9.0E−11 82 H2M3368N 1.3E−10 79 H2M3373N9.0E−10 80 H4H3094P 1.2E−10 85 H4H3095S 1.4E−09 82 H4H3096S 1.8E−10 84H4H3097S E 5 H4H3098S E −13 H4H3099S 9.7E−11 91 H4H3102S E 30 H4H3103S2.4E−10 90 H4H3104S 3.8E−10 89 H4H3105S 1.6E−10 86 H4H3106S 1.7E−10 86H4H3107S 6.6E−11 83 H4H3305N 3.0E−10 86 H4H3310N 4.5E−10 86 H4H3365N3.7E−10 87 H4H3374N 1.2E−10 86 Control I  3.4E−10* 92 *Denotes theaverage IC₅₀ of three separate experiments.

As shown in Table 6, several antibodies of the invention potently blockthe interaction of PDGFR-beta with its natural ligand PDGF-BB, with IC₅₀values ranging from about 7.6 nM (H1M3299N) to about 66 pM (H4H3107S),and certain antibodies enhanced receptor-ligand interactions (e.g.,H4H3097S, H4H3098S and H4H3102S).

B. Receptor/Ligand Blocking Assessed Using a Real-Time Biosensor Assay

The ability of select anti-human PDGFR-beta antibodies to block ligand(PDGF-BB, PDGF-DD and PDGF-AB) binding to human PDGFR-beta was alsoevaluated using a real-time SPR biosensor assay (Biacore 3000).

Briefly, 400RUs of soluble human PDGFR-beta.mFc (SEQ ID NO:338) wascaptured on a Biacore sensor surface derivatized (covalently coupled)with polyclonal rabbit anti-mouse Fc antibody (GE Healthcare LifeSciences, Piscataway, N.J.). The captured surface was saturated with 300nM of selected anti-PDGFR-beta antibodies for 4 min followed by a 30 nMinjection of ligand (PDGF-BB, PDGF-DD or PDGF-AB) for an additional 4min at 25° C. Real-time binding response was monitored throughout thecourse of the assay and was compared to the binding response measuredwhen PDGF ligand was applied over the derivatized captured controlsurface in the absence of captured antibody. Results are illustrated inFIG. 1.

As seen in FIG. 1, all antibodies displayed the ability to block PDGF-BBand PDGF-AB ligands with fewer antibodies enabling efficient blocking ofPDGF-DD when compared to the no antibody control. Of note wereantibodies H4H3094P, H4H3374N, and Control I, which displayed the leastamount of RU response when ligand was applied over the Biacore sensorsurface.

Example 5. Cross-Competition Analysis of Anti-PDGFR-Beta Antibodies

A cross-competition assay was conducted to assess the ability of selectantibodies to compete with one another for binding to human PDGFR-beta.Briefly, soluble human PDGFR-beta.mmh (SEQ ID NO:337), was captured ontoanti-Penta-his Octet sensor tips (ForteBio Corp., Menlo Park, Calif.).Each PDGFR-beta.mmh-coated sensor tip was saturated for 5 min with afirst anti-PDGFR-beta antibody (Mab #1; 50 μg/mL). Next, each sensor tipwas saturated with a solution of a second anti-PDGFR-beta antibody (Mab#2). The real time response of Mab #2 binding to PDGR-beta.mmhpre-complexed with Mab #1 was then monitored. All assays were performedat 25° C. with a flow rate of 1000 rpm on an Octet RED384 biosensor inOctet HBST buffer according to manufacturer's instructions (ForteBioCorp., Menlo Park, Calif.). Results are illustrated in FIG. 2.

Binding responses of less than 0.1 nM are shown in FIG. 2 in black orgray shading and indicate that the corresponding antibody pairs competewith one another for binding to PDGFR-beta. Binding responses greaterthan 0.2 nM (shown in white boxes in FIG. 2) denote antibody pairs thatdo not compete with one another for binding to PDGFR-beta.

The results of this Example indicate that the anti-PDGFR beta antibodiesof the invention can be grouped into two distinct “bins” based onepitope binding characteristics: Bin 1 includes Control I, H4H3365N,H4H3374N, H4H3103S and H4H3094P. Bin 2 includes H4H3099S, H4H3107S,H4H3305N and H4H3310N. The results of this Example suggest that theantibodies of Bin 1 bind to distinct regions on PDGFR-beta than theantibodies of Bin 2.

Example 6. Inhibition of Ligand-Mediated Receptor Activation and MAPKSignaling with Anti-PDGFR-Beta Antibodies

To further characterize anti-PDGFR-beta antibodies of the presentinvention, a bioassay was developed to detect the activation ofPDGFR-beta by two of its known binding ligands, PDGF BB and DD. Theinteraction between PDGFR-beta receptors and its ligands is necessaryfor the induction of diverse cellular processes including proliferation,survival, migration and morphogenesis (Hoch and Soriano, 2003,Development 130:5769-4784). PDGF receptors are receptor tyrosine kinasesand are formed by homo- or hetero-dimerization of alpha and betareceptors upon activation by PDGF BB and DD. Upon activation,auto-phosphorylation is induced and several signal transduction pathwaycascades are triggered, including the Ras-MAPK (mitogen-activatedprotein kinase) pathway.

To detect the activation of the MAPK signal transduction pathway vialigand binding to PDGFR beta, a stable HEK293 cell line was generated toexpress full length human PDGFR-beta along with a luciferase reporter(Serum-Responsive Element [SRE-luciferase]). HEK293/hPDGFR-beta cellswere seeded in a 96-well plate and maintained in low-serum mediacontaining 0.1% FBS overnight. Following incubation, PDGF BB or DD,serially diluted 1:3, was added to cells at concentrations ranging from100 nM to 0.002 nM, to determine dose response. To examine theinhibition of ligand-activated MAPK signaling cascade, antibodies wereserially diluted at 1:3 and added to cells at a concentration rangingfrom 100 nM to 0.002 nM. PDGF BB and DD concentrations remained constantat 250 pM and 400 pM respectively and luciferase activity was detectedafter 5.5 h. PDGF BB and DD activated human PDGFRb with EC₅₀s of0.04-1.11 nM and 0.34-1.82 nM respectively. The antibody concentrationrequired to inhibit 50% of PDGFR-beta-mediated signaling (IC₅₀) wasdetermined for each antibody. Results are summarized in Table 7. (NB=noblocking; Isotype 1=mouse IgG negative control irrelevant antibody;Isotype 2=human IgG negative control irrelevant antibody).

TABLE 7 IC₅₀ Values for Anti-PDGFR-beta Antibodies Blocking PDGF-BB andPDGF-DD Ligand Activation PDGF-BB (250 pM) PDGF-DD (400 pM) AntibodyIC₅₀ (M) IC₅₀ (M) H4H3094P 4.0E−10 3.9E−10 H4H3095S 6.1E−10 8.2E−10H4H3096S 4.5E−10 5.8E−10 H4H3097S NB NB H4H3098S 1.2E−09 1.1E−09H4H3099S 2.1E−10 1.9E−10 H4H3102S 4.1E−09 4.4E−09 H4H3103S 2.0E−102.6E−10 H4H3104S 5.0E−10 3.3E−10 H4H3105S 5.8E−10 5.1E−10 H4H3106S7.4E−10 5.2E−10 H4H3107S 1.7E−10 2.4E−10 H1M3299N 5.6E−10 4.2E−10H1M3305N 8.5E−09 1.9E−10 H1M3310N 2.3E−08 2.8E−10 H1M3361N 6.8E−098.4E−11 H2M3363N 7.5E−09 1.9E−10 H2M3365N 7.9E−09 1.1E−10 H2M3368N1.8E−10 1.7E−10 H2M3373N 7.0E−11 9.2E−11 H1M3374N 3.1E−10 2.1E−10H4H3305N 5.0E−10 4.8E−10 H4H3310N 6.8E−10 6.6E−10 H4H3365N 2.3E−103.7E−10 H4H3374N 1.3E−10 1.5E−10 Control I 1.8E−10 1.8E−10 Isotype 1 NBNB Isotype 2 NB NB

As shown in Table 7, several of the anti-PDGFR-beta antibodies of thepresent invention potently blocked ligand-dependent PDGFR-betaactivation, with IC₅₀s in the sub-nanomolar range. Additionally, bothmouse IgG (isotype 1) and human IgG (isotype 2) negative controls didnot block ligand activation of the receptor.

Example 7. Internalization of Anti-PDGFR-Beta Antibodies onPDGFR-Beta-Expressing Cells

To study antibody mediated receptor internalization, experiments wereperformed using cells engineered to express human PDGFR-beta(HEK293/SRE-luc/PDGFRb cells). Briefly, 20,000 HEK293/SRE Luc/PDGFRbcells/well were plated overnight in full media (10% FBS, Pen/Strep/Glut,NEAA, and G418 in DMEM) and stained with anti-PDGFR-beta antibodies at10 pg/ml for 30 mins at 4° C. Cells were washed twice and stained withDylight 488 conjugated Fab goat anti-human IgG secondary antibody (10ug/mL; Jackson ImmunoResearch Laboratories, West Grove, Pa.) for 30 minsat 4° C. Next, cells were incubated at 37° C. for 2 hours to allowreceptor internalization. Alexa-488 fluorescence was quenched byincubating washed cells with anti-Alexa fluor 488 (Invitrogen Corp.,Carlsbad, Calif.) for 45 mins at 4° C. to differentiate surface-boundantibodies from the internalized antibodies. Images were taken withImageXpress Micro XL (Molecular Devices LLC, Sunnyvale, Calif.) and spotanalysis was performed using Columbus software (Perkin Elmer, Waltham,Mass.). Relative internalization was calculated by comparing thequenched staining (i.e. internalized antibody) of each antibody to thatof the Control 1 antibody. Results are summarized in Table 8.

TABLE 8 Internalization of Select Anti-PDGFR-beta Antibodies PercentInternalization Antibody (Relative to Control I) H4H3094P 77% H4H3099S88% H4H3103S 87% H4H3107S 92% H4H3305N 79% H4H3310N 66% H4H3365N 65%H4H3374N 81% Isotype Ctrl  4% Control I 100% 

As shown in Table 8, all anti-PDGFR-beta antibodies studied showedrobust internalization in this assay format, reflecting the potentialability of the antibodies to effectively target PDGFR-beta-expressingcells in various therapeutic contexts.

Example 8. Anti-PDGFR-Beta Antibodies Bind Within Distinct Domains onPDGFR-Beta

The extracellular portion of PDGFR-beta consists of 5 Ig-like C2-typedomains, referred to as D1-D5. D1 through D3 are required for highaffinity ligand binding. In this Example, experiments were conducted todetermine which extracellular domain(s) certain anti-PDGFR-betaantibodies of the invention interact with.

For this experiment, four different PDGFR-beta extracellular domainconstructs were used: D1 (SEQ ID NO:342), D1-D2 (SEQ ID NO:343), D1-D3(SEQ ID NO:344), and D1-D4 (SEQ ID NO:345), as well as full-lengthPDGFR-beta. Four different anti-PDGFR-beta antibodies were tested forbinding to the various constructs by surface plasmon resonance(Biacore). Briefly, 150-200 RU's of anti-PDGFR beta antibody wascaptured via an anti-human Fc CM5 chip. Next, the individual domainconstructs, or full-length PDGFR beta, was applied over theantibody-bound surface at a concentration of 50 nM. The ability of thevarious antibodies to bind to the various domain constructs wasmeasured. Results are shown in Table 9. (−)=No binding observed;(+)=Binding observed; ND=Not determined.

TABLE 9 Observed Binding of Selected Anti-PDGFR-beta Antibodies toPDGFR-beta Domains and Full-length PDGFR-beta Protein PredictedPDGFR-beta Domains Full-Length Domain of Antibody D1 D1-2 D1-3 D1-4PDGFR beta Binding H4H3094P − + + + + 2 H4H3099S − − − − + ND H4H3305N −− − − + ND H4H3374N − + + + + 2

As summarized in Table 9, all antibodies bound to full-lengthPDGFR-beta. Two antibodies, H4H3094P and H4H3374N, were determined tobind to domain 2. Interestingly these two antibodies are also ligandblockers based on the ELISA immunoassay, confirming that domain 2 isimportant for ligand (PDGF-BB) binding. The two other exemplaryantibodies tested, H4H3099S and H4H3305N, did not bind to any of thedomain constructs, suggesting that these antibodies may need the aminoacids between domains 4 and 5 and/or domain 5 itself for high affinitybinding.

Example 9. Anti-PDGFR-Beta Antibodies Deplete Pericytes in an In VivoRetinal Model

Two exemplary anti-PDGFR beta antibodies, H4H3374N and H4H3094P, weretested in an in vivo retinal pericyte depletion model. Pericytes aresmooth-muscle-like cells that express PDGFR-beta. PDGF-B, expressed onendothelial cells, plays a role in the recruitment of pericytes to newlyforming vessels, thus promoting angiogenesis and the establishment ofvascular architecture. However, the interaction between pericytes andthe endothelium, and PDGF-B/PDGFR-beta signaling, is disrupted duringpathogenic angiogenesis, contributing to uncontrolled vessel formation.In diseases of the eye, this neovascularization can lead to visualmorbidity and blindness.

In a first experiment, humanized PDGFR-beta mouse pups were injectedsubcutaneously (s.c.) with 3 mg/kg H4H3374N, H4H3094P, control I (2C5)or human Fc (hFc) to see the effect of blocking PDGF-B/PDGFR-betasignaling in newly forming vasculature. Briefly, post-natal day 2 (P2)humanized PDGFR-beta pups were injected subcutaneously with 3 mg/kg ofhFc control or PDGFR-beta antibody. On post-natal day 5, pups weresacrificed. Both eyes were collected and fixed in 4% P.F.A for 1 h. Eyeswere washed 3× with PBS and retinas were dissected removing hyaloidvessels. Retinas were stained O/N at room temp with a rabbit anti-NG2chondroitin sulfate primary antibody prepared in antibody dilution serum(ADS; 1% BSA in 0.05% Triton-X-100 in PBS). After incubation, allretinas were washed 3× for 15 min in PBS and then stained 0/N at 4° C.with fluorescein labeled Griffonia Simplicifolia lectin and a goatanti-rabbit alexa 594 labeled secondary prepared in ADS. Afterincubation, all retinas were again washed 3× for 15 min in PBS. Retinaswere flat-mounted on slides and cover-slipped using Fluoromount-G™without DAPI.

Retinas were imaged using a Nikon 80i fluorescent microscope. Imageswere analyzed using Adobe Photoshop and Fovea. The average NG2 positivearea, normalized to the hFc, was measured for each treatment group. Bothimaging and analysis were performed in a blinded fashion. Statisticalanalysis was done using one-way ANOVA in prism software. Results aresummarized in Tables 10-11.

TABLE 10 Reduction in NG2 Positive Retinal Area Post Treatment with 3mg/kg H4H3374N, Control I or hFc Normalized NG2 Area Relative to hFc NhFc Control I (2C5) H4H3374N 1 1.0 1.00 0.13 2 1.0 0.48 0.25 3 1.0 0.760.14 4 1.0 0.68 0.15 5 1.0 0.64 — Avg 1.0 0.71 0.17

TABLE 11 Reduction in NG2 Positive Retinal Area Post Treatment with 3mg/kg H4H3094P, Control I or hFc Normalized NG2 Area Relative to hFc NhFc Control I (2C5) H4H3094P 1 1.0 0.88 0.79 2 1.0 0.85 0.61 3 1.0 0.850.37 4 1.0 0.87 0.66 5 1.0 0.88 0.83 Avg 1.0 0.86 0.65

As shown in Tables 10-11, the average retinal NG2 positive area wasdecreased in mice treated with the anti-PDGFR-beta antibodies comparedto the hFc. The NG2 positive area was significantly decreased (p<0.001)for antibodies H4H3374N and H4H3094P relative to hFc. Furthermore,H4H3374N displayed the greatest reduction in NG2 positive area whencompared to both H4H3094P and the Control I antibody.

In a separate set of experiments, C57Bl/6 mouse pups were injectedsubcutaneously (SC) at P2 with an anti-mouse PDGFR-beta antibody “mAb39”(having the variable regions of the antibody referred to as APB5, seeUemura et al., J. Clin. Invest. 2002; 110(11):1619-1628) at doses of 50mg/kg, 25 mg/kg, 12.5 mg/kg, or 6.25 mg/kg, or with Fc at 50 mg/kg as acontrol (Study 1). The effect on pericyte coverage was assessed at P5using a rabbit anti-NG2 chondroitin sulfate proteoglycan 4 primaryantibody. In the developing retinal vessels, all doses of mAb39 12.5mg/kg inhibited blood vessel pericyte coverage.

In another study (Study 2), P2 pups were injected SC with 25 mg/kg ofmAb39 or control. Retinas were collected at P5 and stained withGriffonia simplicifolia lectins (“GS Lectin 1,” Vector Labs). At a 25mg/kg dose, mAb39 moderately decreased vascularized retinal areas andvessel density compared to controls.

In a separate set of experiments (Study 3), left eyes of pups wereinjected intravitreally (IVT) with 5 pg (0.5 μl) of mAb39 or control atP4 and collected at P6. A single intravitreal anti-PDGFR-beta antibodyadministration almost completely depleted mural cells and producedmarked effects on retinal vascular differentiation and morphology, e.g.,irregular blood vessel caliber. Additional experiments were conducted toinvestigate the effect of PDGFR-beta neutralization in the eyes of adultmice. In particular, left eyes of adult mice were injected IVT withmAb39 (5 μg or 10 μg) or control (5 μg or 10 μg). Eyes were collected 48hrs later and stained with anti-NG2 and GS Lectin I. In adult mice,mAb39 produced no evidence of any pericyte loss or any vascularmorphological changes.

These studies collectively demonstrate that selective pharmacologicalneutralization of PDGFR-beta is effective in promoting pericytedepletion and contributes to changes in vascular morphology and growthin developing retinal neovessels. In contrast, this same inhibition doesnot appear to have any effect on mature pericytes and vessels in theestablished vasculature in the adult mouse retina.

Example 10. A Phase 1 Clinical Trial of a Combination FormulationComprising an Anti-PDGFR-Beta Antibody and a VEGF Antagonist in Patientswith Age-Related Macular Degeneration

Study Overview

A phase 1 clinical trial is conducted to test the safety of ananti-PDGFR-beta antibody of the invention delivered by intravitrealinjection in patients with neovascular age-related macular degeneration(AMD) in conjunction with intravitreal (IVT) aflibercept. The amino acidsequence of aflibercept (also known as VEGFR1R2-FcΔC1(a)), as well asthe nucleic acid sequence encoding the same, are set forth, e.g., in WO2012/097019, the disclosure of which is incorporated by reference hereinin its entirety.

The primary objective of this study is to investigate the safety ofintravitreal (IVT) anti-PDGFR-beta antibody in patients with neovascularAMD. The secondary objectives are to explore the anatomic effects of IVTanti-PDGFR-beta on corneal neovascularization (CNV) in patients withneovascular AMD, and to determine the pharmacokinetics ofanti-PDGFR-beta and aflibercept in humans. Another objective of thisstudy is to determine the presence of antibodies against theanti-PDGFR-beta antibody and/or aflibercept in subjects treated withthese agents.

Target Population

The target population for this study is men and women aged 50 years andolder with neovascular AMD. Approximately 3-6 patients will be enrolledin four planned cohorts. A total of 15-24 patients is planned. Sixpatients will be enrolled at the maximum tolerated dose (MTD), ifidentified, or the highest dose level.

Key Inclusion/Exclusion Criteria

The key inclusion criteria for this study are as follows: (1) men orwomen 50 years of age or older; and (2) active subfoveal CNV secondaryto AMD, including juxtafoveal lesions that affect the fovea as evidencedby FA in the study eye.

The key exclusion criteria are as follows: (1) IVT anti-VEGF therapy inthe study eye within 8 weeks of the start of the study (Day 1); (2) anyprior treatment with PDGF or PDGFR inhibitors; (3) intraocular pressuregreater than or equal to 25 mmHg in the study eye; (4) evidence ofinfectious blepharitis, keratitis, scleritis, or conjunctivitis ineither eye; (5) any intraocular inflammation/infection in either eyewithin 3 months of the screening visit; (6) current irisneovascularization, vitreous hemorrhage, or tractional retinaldetachment visible at the screening assessments in the study eye; (7)evidence of CNV due to any cause other than AMD in either eye; (8)evidence of diabetic retinopathy or diabetic macular edema in eithereye; (9) inability to obtained photographs, FA or OCT to document CNV,e.g., due to media opacity, allergy to fluorescein dye or lack of venousaccess; and (10) systemic (IV) anti-VEGF administration within 6 weeksof Day 1.

Study Design

Patients will be assessed for study eligibility at the screening visit,up to 2 weeks before Day 1/baseline (Visit 2). At the Day 1/baseline(Visit 2), patients will undergo safety assessments prior to receivingthe first dose of study drug.

Eligible patients will be enrolled into the current cohort that is opento enrollment. The initial cohort will receiveanti-PDGFR-beta/aflibercept (coformulated at 0.2 mg: 2 mg). On Day 1 andDay 29 (±3 days), patients will receive an injection ofanti-PDGFR-beta/aflibercept.

The dose of anti-PDGFR-beta/aflibercept will be escalated based onsafety and tolerability assessed during the previous cohort (startingfrom the first patient, first dose to 2 weeks following the lastpatient's second dose in that cohort, or approximately Week 6). Also,the first patient enrolled in each cohort will be observed for at least1 week after the first dose before additional patients are dosed.Escalation to the next dose cohort will occur once the data have beenreviewed. Intra-patient dose escalation will not be permitted.

Patients will be evaluated at study visits for ocular and systemicsafety (including ophthalmic exam, laboratory assessments, etc.) andefficacy (OCT, FA/FP, CNV area, classic CNV size, total lesion size,macular volume, imaging, and BCVA using the 4-meter ETDRS protocol) andwill be followed to Week 24.

Study Drug Treatments

Four different anti-PDGFR-beta/aflibercept co-formulations will beadministered to patients. The co-formulations are summarized in Table12.

TABLE 12 Anti-PDGFR-beta Co-Formulation Antibody Aflibercept 1 0.2 mg 2mg 2 0.5 mg 2 mg 3   1 mg 2 mg 4   3 mg 2 mg

Each formulation will consist of 10 mM sodium phosphate, pH 6.2, 0.03%(w/v) polysorbate 20, 5% (w/v) sucrose, and 40 mM sodium chloride.

The various anti-PDGFR-beta/aflibercept co-formulations will bedelivered via IVT injection and the injection volume will be 50 μl. Asnoted above, patients will receive two separate administrations of theco-formulation. The first administration will be on Day 1, and thesecond administration will be on Day 29.

Primary and Secondary Endpoints

The primary endpoint of the study is safety of study drug. Secondaryendpoints are: (1) change in central retinal thickness from baseline(measured by OCT) at Week 8 and Week 12; (2) proportion of patients withcomplete resolution of retinal fluid (measured by OCT) at Week 8 andWeek 12; (3) change in CNV area from baseline (measured by OCT) at Week8 and Week 12; (4) change in CNV size from baseline (measured by FA) atWeek 8 and Week 12; (5) change in area of leakage from baseline(measured by FA) at Week 8 and Week 12; (6) change in BCVA frombaseline; and (7) pharmacokinetics and development of anti-drugantibodies.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof that specifically binds human platelet derived growth factorreceptor beta (PDGFR-beta), wherein the antibody or antigen-bindingfragment thereof comprises: a heavy chain variable region (HCVR), and alight chain variable region (LCVR), the HCVR comprising three heavychain complementarity determining regions (HCDR1, HCDR2 and HCDR3)comprising SEQ ID NOs: 132, 134 and 136, respectively; and the LCVRcomprising three light chain complementarity determining regions (LCDR1,LCDR2 and LCDR3) comprising SEQ ID NOs: 140, 142 and 144, respectivelywherein the antibody or antigen-binding fragment thereof differs by onlya single amino acid residue in one of the HCVR of SEQ ID NO:130 or theLCVR of SEQ ID NO:138, with the proviso that each of the sequences SEQID NOs: 132, 134, 136, 140, 142 and 144 remain unchanged.
 2. Theisolated antibody or antigen-binding fragment of claim 1, whereinantibody or antigen-binding fragment thereof binds PDGFR-beta with abinding dissociation equilibrium constant (K_(D)) of less than about 200pM as measured in a surface plasmon resonance assay at 37° C.
 3. Anisolated antibody or antigen-binding fragment thereof that specificallybinds monomeric human platelet derived growth factor receptor beta(PDGFR-beta) with a binding dissociation equilibrium constant (K_(D)) ofless than about 200 pM as measured in a surface plasmon resonance assayat 37° C., wherein the antibody or antigen-binding fragment thereofcomprises: a heavy chain variable region (HCVR), and a light chainvariable region (LCVR), the HCVR comprising three heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3) comprisingSEQ ID NOs: 132, 134 and 136, respectively; and the LCVR comprisingthree light chain complementarity determining regions (LCDR1, LCDR2 andLCDR3) comprising SEQ ID NOs: 140, 142 and 144, respectively wherein theantibody or antigen-binding fragment thereof differs by only a singleamino acid residue in one of the HCVR of SEQ ID NO:130 or the LCVR ofSEQ ID NO:138, with the proviso that each of the sequences SEQ ID NOs:132, 134, 136, 140, 142 and 144 remain unchanged.
 4. An isolatedantibody or antigen-binding fragment thereof that specifically bindshuman platelet derived growth factor receptor beta (PDGFR-beta), whereinthe antibody or antigen-binding fragment thereof comprises: a heavychain variable region (HCVR), and a light chain variable region (LCVR),the HCVR comprising three heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) comprising SEQ ID NOs: 132, 134 and136, respectively; and the LCVR comprising three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) comprisingSEQ ID NOs: 140, 142 and 144, respectively wherein the antibody orantigen-binding fragment thereof differs by only a single amino acidresidue in one of the HCVR of SEQ ID NO:130 or the LCVR of SEQ IDNO:138, with the proviso that each of the sequences SEQ ID NOs: 132,134, 136, 140, 142 and 144 remain unchanged and the antibody orantigen-binding fragment is bioequivalent to an antibody orantigen-binding fragment which comprises SEQ ID NO:130 and SEQ IDNO:138.